Tumor hypoxia has emerged as one of the most important drivers of metastasis, therapy resistance and poor clinical outcome in many cancers, including prostate cancer. Current treatment options for either localized or metastasized prostate tumors do not address hypoxic cells. Because of the positive role of hypoxia on metastasis and therapy resistance, there is a critical need for the identification, characterization, and development of therapies that target hypoxic prostate tumor cells. Mammalian orthoreovirus (MRV) is a potent oncolytic virus that has been shown to be safe and effective in vitro, in animal models, and in human clinical trials against a number of cancers, including prostate cancer. Our long-term goals are to identify and characterize genetic pathways modulated by MRV to provide a critical biological knowledge base that supports and informs the utility of MRV as a cancer therapy. In preliminary results, we have found that MRV infection of hypoxic prostate tumor cells leads to downregulation of the regulatory subunit (HIF-1?) of the master transcriptional regulator of hypoxia, HIF-1, via proteasome-mediated degradation and translational inhibition. We have further shown that MRV-induced degradation of HIF-1? occurs via a receptor for activated kinase C (RACK1)-dependent mechanism. Moreover, we found that MRV infection also downregulates androgen receptor (AR) expression and overrides increases in AR activity induced by hypoxia in prostate tumor cells. Based on these data, our central hypothesis is that MRV infection and modulation of HIF-1? interferes with AR expression and will enhance AR signaling inhibitor therapy. In this proposal, we will identify the mechanism of MRV-induced HIF-1? downregulation in hypoxic prostate tumor cells by elucidating the impact of MRV infection on HIF-1? interaction with regulatory proteins RACK1 and HSP90, determining the mechanism of MRV-induced HIF-1? translation inhibition, and investigating viral proteins involved in HIF-1? downregulation. We will also examine the impact of MRV-induced HIF-1? downregulation on AR in infected cells, elucidate the mechanism of, and viral proteins involved in, MRV-induced AR downregulation, and determine if MRV infection similarly downregulates AR mutants associated with progression to androgen-independence. Finally, we will examine the impact of dual MRV and AR signaling inhibitors on HIF-1? and AR expression and cell death. As MRV therapy for cancer has advanced to clinical trials, these studies are highly significant because they will provide an underlying mechanistic picture of how MRV infection alters tumor cell signaling at multiple progressive stages of PCa, and innovative because they will substantially expand our understanding of the molecular events underlying MRV modulation of key pathways involved in PCa progression and chemotherapy resistance.